Pharmaceutical compositions containing anti-RANKL-NGF bispecific antibodies

Abstract

The present disclosure relates to pharmaceutical compositions comprising anti-RANKL-NGF bispecific antibodies. Specifically, pharmaceutical compositions related to the present disclosure comprise an anti-RANKL-NGF bispecific antibody and a buffering agent.

Description

[Technical Field]

[0001] This application claims priority from a Chinese patent application (application number 202211438239.8, filing date November 16, 2022).

[0002] The present disclosure belongs to the field of drug formulations, and specifically relates to pharmaceutical compositions comprising anti-RANKL-NGF bispecific antibodies and their use as medicines. [Background technology]

[0003] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0004] Bone metastasis is a common symptom of many cancers in their late stages. The pathogenesis of bone metastasis is as follows: primary tumor cells shed and enter the blood circulation, forming circulating tumor cells. These circulating tumor cells then metastasize to bone sites, forming metastatic tumor cells, and then remaining dormant in the bone microenvironment. This process can continue for several years until the surviving tumor cells gradually adapt to the bone microenvironment and become activated from a dormant state to a proliferative state (Mantyh, PW, Curr Opin Support Palliat Care, 2014.8(2):pp.83-90). Tumor cell proliferation affects the bone microenvironment and promotes osteoclast formation, which accelerates bone resorption and releases various cytokines, further promoting tumor cell proliferation, creating a vicious cycle (Quayle, L., Current Cancer Drug Targets, 2015.15(6):pp.469-480). The continued proliferation of tumor cells leads to the formation of multiple metastatic sites, causing various problems for patients, such as bone damage and pain (Gartland, A., Journal of Bone Oncology, 2016.5(3):p100-103).

[0005] Statistics show that common cancers that cause bone metastasis include myeloma, kidney cancer, melanoma, bladder cancer, thyroid cancer, lung cancer, breast cancer, and prostate cancer (Clezardin, P., Joint Bone Spine, 2017. 84(6): p677-684; Fidler, M.M. Fidler, Scandinavian Journal of Public Health, 2018. 46(1): p27-36).

[0006] In normal bone tissue, osteoblasts and osteoclasts maintain a relative equilibrium to maintain normal bone growth and development. However, the presence of cancer cells disrupts this equilibrium. Cancer cells in the bone microenvironment release a series of cytokines that stimulate osteoblasts and osteocytes to secrete large amounts of Receptor Activator of Nuclear Factor-κB Ligand (RANKL). RANKL acts on the RANK receptor to promote osteoclast formation and increase bone resorption. Osteolysis due to bone resorption further releases other growth factors, promoting cancer cell proliferation. This creates a circulation that favors cancer cell metastasis (Body, JJ, Expert Rev Anticancer Ther, 2012.12(3):p307-322).

[0007] Nerve growth factor (NGF) is a nerve growth factor. The NGF signaling pathway mediates nervous system growth and development and pain signal transduction. Two types of NGF receptors are known to exist on the cell surface: TrKA, a high-affinity receptor, and p75NTR, a low-affinity receptor. NGF acts on TrkA to simultaneously activate the Ras and PI3K pathways, promoting cell survival and nerve growth. On the other hand, NGF acts on p75NTR to promote cell apoptosis. Furthermore, the PI3K pathway activates TRPV1 phosphorylation, activating ion channels to generate action potentials and transmit pain neuron signals (Kumar, V. and Mahal, B.A., Journal of Pain Research, 2012.5:279-287).

[0008] Data show that monoclonal antibodies targeting NGF and RANKL have some therapeutic effects on bone damage and pain caused by bone metastasis (Body JJ. Expert Rev Anticancer Ther. 2012.12(3):p307~322. Sopata M., et al. 2015.156(9):p1703~1713). Summary of the Invention

[0009] The present disclosure provides a pharmaceutical composition comprising an anti-RANKL-NGF bispecific antibody, which has therapeutic activity and also has the advantage of good stability.

[0010] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-RANKL-NGF bispecific antibody and a buffer, wherein the anti-RANKL-NGF bispecific antibody comprises a first antigen-binding domain that specifically binds to RANKL and a second antigen-binding domain that specifically binds to NGF, and the buffer is an acetate buffer, a histidine buffer, or a phosphate buffer.

[0011] In some embodiments, the buffer is an acetic acid-sodium acetate buffer, a histidine-histidine hydrochloride buffer, or a citrate-disodium hydrogen phosphate buffer.

[0012] In some embodiments, the buffer is an acetic acid-sodium acetate buffer or a histidine-histidine hydrochloride buffer.

[0013] In some specific embodiments, the buffer is an acetic acid-sodium acetate buffer.

[0014] In some embodiments, the pharmaceutical composition is any one of the above, wherein the pH of the pharmaceutical composition is 4.2 to 7.8. In some embodiments, the pH of the pharmaceutical composition is 4.2 to 7.0. In some embodiments, the pH of the pharmaceutical composition is 4.2 to 5.4. In some embodiments, the pH of the pharmaceutical composition is 4.6 to 5.4. In some embodiments, the pH of the pharmaceutical composition is about 4.6. In some embodiments, the pH of the pharmaceutical composition is about 4.8. In some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is about 5.2. In some embodiments, the pH of the pharmaceutical composition is about 5.4. When a point value is referred to in this disclosure, it should be understood that the point value includes a margin of error. This margin of error is due to factors such as laboratory environment, operator manipulation, equipment, methodology, and measurement error. Taking pH as an example, a measured value of about 5.0 should be understood to include a margin of error. As an example, when measuring a formulation with an industrial pH meter, "about 5.0" indicates 5.0±0.2 (ie, a pH of 4.8 to 5.2).

[0015] In some embodiments, the pH of the pharmaceutical composition is 4.6 to 6.6. In some embodiments, the pH of the pharmaceutical composition is 4.6 to 5.8. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.8.

[0016] In some embodiments, the pH of the pharmaceutical composition is 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8, or any range therebetween. In some embodiments, the pH of the pharmaceutical composition is 4.6. In some embodiments, the pH of the pharmaceutical composition is 4.8. In some embodiments, the pH of the pharmaceutical composition is 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.2. In some embodiments, the pH of the pharmaceutical composition is 5.4.

[0017] Typically, the pH of the pharmaceutical composition obtained by replacing the buffer is approximately the same as the pH of the buffer. At the same time, as known to those skilled in the art, although pH drift may occur during the drug formulation process, the pH drift of the drug formulation is generally very small (e.g., within ±0.3). In some embodiments, the pH drift of the drug formulation is within ±0.2. In some embodiments, the pH drift of the drug formulation is within ±0.1.

[0018] In some embodiments, in any one of the above pharmaceutical compositions, the anti-RANKL-NGF bispecific antibody has a concentration of 1 to 150 mg / mL. In some embodiments, the anti-RANKL-NGF bispecific antibody has a concentration of 1 to 100 mg / mL. In some embodiments, the anti-RANKL-NGF bispecific antibody has a concentration of 10 to 80 mg / mL. In some embodiments, the anti-RANKL-NGF bispecific antibody has a concentration of 20 to 80 mg / mL. In some embodiments, the anti-RANKL-NGF bispecific antibody has a concentration of 20 to 77 mg / mL. In some embodiments, the anti-RANKL-NGF bispecific antibody has a concentration of 20 to 70 mg / mL. In some embodiments, the anti-RANKL-NGF bispecific antibody has a concentration of 50 to 77 mg / mL. In some embodiments, the anti-RANKL-NGF bispecific antibody has a concentration of 56 to 84 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 63 to 77 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is about 77 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is about 70 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is about 50 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is about 20 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 56 mg / mL, 60 mg / mL, 63 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 77 mg / mL, 80 mg / mL, 84 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, or 150 mg / mL, or any range therebetween. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 77 mg / mL.In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 70 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 50 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 20 mg / mL.

[0019] In some embodiments, the pharmaceutical composition according to any one of the above, further comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from the group consisting of poloxamer (e.g., poloxamer 188), polysorbate (e.g., polysorbate 20, polysorbate 80), poloxamer, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleic sulfobetaine, stearic sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleic sarcosine, stearic sarcosine, linoleic betaine, and myristyl betaine. The surfactant may be selected from the group consisting of methyl betaine, cetyl betaine, lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, and the like. In some embodiments, the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20. In some embodiments, the surfactant is polysorbate 80.

[0020] In some embodiments, in the pharmaceutical composition described above, the surfactant has a concentration of 0.01 to 1.0 mg / mL. In some embodiments, the surfactant has a concentration of 0.01 to 0.8 mg / mL. In some embodiments, the surfactant has a concentration of 0.01 to 0.6 mg / mL. In some embodiments, the surfactant has a concentration of 0.01 to 0.4 mg / mL. In some embodiments, the surfactant has a concentration of 0.01 to 0.2 mg / mL. In some embodiments, the surfactant has a concentration of 0.1 to 0.4 mg / mL. In some embodiments, the surfactant has a concentration of 0.1 to 0.2 mg / mL. In some embodiments, the surfactant has a concentration of 0.05 to 0.15 mg / mL. In some embodiments, the surfactant has a concentration of 0.08 to 0.12 mg / mL. In some embodiments, the surfactant has a concentration of 0.09 to 0.11 mg / mL. In some embodiments, the surfactant concentration is about 0.4 mg / mL. In some embodiments, the surfactant concentration is about 0.2 mg / mL. In some embodiments, the surfactant concentration is about 0.1 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1.0 mg / mL, or any range between these values. In some embodiments, the surfactant concentration is 0.4 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL. In some embodiments, the concentration of the surfactant is 0.1 mg / mL.

[0021] In some embodiments, the surfactant is about 0.1 mg / mL polysorbate 80. In some embodiments, the surfactant is 0.1 mg / mL polysorbate 80.

[0022] In some embodiments, the pharmaceutical composition described in any one of the above further comprises a stabilizer. In some embodiments, the stabilizer is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), an amino acid (including proline, arginine, glycine, cysteine, histidine, etc.), or a salt (sodium chloride, potassium chloride, calcium chloride, etc.). In some embodiments, the stabilizer is one or more selected from the group consisting of proline, sucrose, trehalose, sorbitol, arginine, glycine, and sodium chloride. In some embodiments, the stabilizer is proline, sucrose, or sodium chloride. In some embodiments, the stabilizer is an amino acid. In some embodiments, the stabilizer is proline.

[0023] In some embodiments, in the pharmaceutical composition described above, the stabilizer concentration is 1 to 300 mM. In some embodiments, the stabilizer concentration is 25 to 290 mM. In some embodiments, the stabilizer concentration is 25 to 250 mM. In some embodiments, the stabilizer concentration is 210 to 270 mM. In some embodiments, the stabilizer concentration is 216 to 264 mM. In some embodiments, the stabilizer concentration is 228 to 252 mM. In some embodiments, the stabilizer concentration is about 240 mM. In some embodiments, the concentration of the stabilizer is 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 216 mM, 220 mM, 228 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 252 mM, 260 mM, 264 mM, 270 mM, 280 mM, 290 mM, or 300 mM, or any range between these point values. In some embodiments, the concentration of the stabilizer is 240 mM.

[0024] In some embodiments, the stabilizer is about 240 mM proline. In some embodiments, the stabilizer is 240 mM proline.

[0025] In some embodiments, the stabilizer is a sugar selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol (also known as sorbitol), mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, maltitol, lactitol, and iso-maltulose. In some embodiments, the stabilizer is one or more selected from the group consisting of sucrose, trehalose, sorbitol, arginine, glycine, and sodium chloride. In some embodiments, the stabilizer is a non-reducing disaccharide. In some embodiments, the stabilizer is trehalose or sucrose. In some embodiments, the stabilizer is sucrose.

[0026] In some embodiments, the stabilizer is 10-100 mg / mL sucrose. In some embodiments, the stabilizer is 30-80 mg / mL sucrose. In some embodiments, the stabilizer is 50-80 mg / mL sucrose. In some embodiments, the stabilizer is 70-80 mg / mL sucrose. In some embodiments, the stabilizer is 60-90 mg / mL sucrose. In some embodiments, the stabilizer is 67.5-82.5 mg / mL sucrose. In some embodiments, the stabilizer is about 75 mg / mL sucrose. In some embodiments, non-limiting examples of stabilizer concentrations include 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 65 mg / mL, 67.5 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 82.5 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, and any range between these values. In some embodiments, the stabilizer is 75 mg / mL of sucrose.

[0027] In some embodiments, the stabilizer is a salt. In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizer is 0.64% (w / v) to 0.96% (w / v) sodium chloride. In some embodiments, the stabilizer is 0.72% (w / v) to 0.88% (w / v) sodium chloride. In some embodiments, the stabilizer is about 0.8% (w / v) sodium chloride. In some embodiments, the stabilizer is 0.8% (w / v) sodium chloride.

[0028] In some embodiments, in the pharmaceutical composition described above, the buffering agent has a concentration of 5 to 100 mM. In some embodiments, the buffering agent has a concentration of 10 to 50 mM. In some embodiments, the buffering agent has a concentration of 10 to 30 mM. In some embodiments, the buffering agent has a concentration of 10 to 20 mM. In some embodiments, the buffering agent has a concentration of 16 to 24 mM. In some embodiments, the buffering agent has a concentration of 18 to 22 mM. In some embodiments, the buffering agent has a concentration of about 20 mM. In some embodiments, the buffering agent has a concentration of about 10 mM. In some embodiments, the buffer concentration is 5 mM, 10 mM, 15 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, and any range between these values. In some embodiments, the buffer concentration is 20 mM. In some embodiments, the buffer concentration is 10 mM.

[0029] In some embodiments, the buffer is about 10 mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is about 20 mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 10 mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 20 mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 10 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 10 mM citrate-disodium hydrogen phosphate buffer.

[0030] In some embodiments, the pharmaceutical composition is any one of the above, wherein the anti-RANKL-NGF bispecific antibody comprises at least one first antigen-binding domain that specifically binds to RANKL and at least one second antigen-binding domain that specifically binds to NGF.

[0031] In some embodiments, the pharmaceutical composition is any one of the above, wherein the anti-RANKL-NGF bispecific antibody comprises two first antigen-binding domains that specifically bind to RANKL and two second antigen-binding domains that specifically bind to NGF.

[0032] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the anti-RANKL-NGF bispecific antibody has the structure shown in FIG.

[0033] In some embodiments, in the pharmaceutical composition according to any one of the above, the first antigen-binding domain that specifically binds to RANKL in the anti-RANKL-NGF bispecific antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and The light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:4, LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0034] In some embodiments, the pharmaceutical composition is any one of the above, wherein in the anti-RANKL-NGF bispecific antibody, the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0035] In some embodiments, in the pharmaceutical composition according to any one of the above, wherein the second antigen-binding domain that specifically binds to NGF in the anti-RANKL-NGF bispecific antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; and The light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:18, LCDR2 comprising the amino acid sequence of SEQ ID NO:19, and LCDR3 comprising the amino acid sequence of SEQ ID NO:20.

[0036] In some embodiments, in the pharmaceutical composition according to any one of the above, wherein the second antigen-binding domain that specifically binds to NGF in the anti-RANKL-NGF bispecific antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21; and / or The light chain variable region comprises the amino acid sequence of SEQ ID NO:22.

[0037] In some embodiments, the pharmaceutical composition of any one of the above-mentioned embodiments, wherein in the anti-RANKL-NGF bispecific antibody, the second antigen-binding domain that specifically binds to NGF comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 24 and the light chain comprises the amino acid sequence of SEQ ID NO: 25.

[0038] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the anti-RANKL-NGF bispecific antibody: (i) the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) the second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; and The light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:18, LCDR2 comprising the amino acid sequence of SEQ ID NO:19, and LCDR3 comprising the amino acid sequence of SEQ ID NO:20.

[0039] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the anti-RANKL-NGF bispecific antibody: (i) the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; (ii) The second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22.

[0040] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the anti-RANKL-NGF bispecific antibody: (i) the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) the second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; and The light chain variable region comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 18, LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, and the anti-RANKL-NGF bispecific antibody has the structure shown in Figure 1.

[0041] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the anti-RANKL-NGF bispecific antibody: (i) the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; (ii) the second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22, and the anti-RANKL-NGF bispecific antibody has the structure shown in Figure 1.

[0042] In some embodiments, the pharmaceutical composition is any one of the above, wherein the anti-RANKL-NGF bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 31.

[0043] In some embodiments, the anti-RANKL-NGF bispecific antibody comprises two first chains of identical sequence and two second chains of identical sequence, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 31.

[0044] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 150 mg / mL of the anti-RANKL-NGF bispecific antibody; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 1 to 300 mM of a stabilizer; (d) 5 to 100 mM of a buffering agent, and the pH of the pharmaceutical composition is 4.2 to 7.0.

[0045] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 150 mg / mL of the anti-RANKL-NGF bispecific antibody; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 1 to 300 mM proline; (d) 5 to 100 mM of a buffering agent, and the pH of the pharmaceutical composition is 4.2 to 7.0.

[0046] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 10 to 80 mg / mL of the anti-RANKL-NGF bispecific antibody; (b) 0.01 to 0.6 mg / mL of polysorbate 20 or polysorbate 80; (c) 25 to 290 mM proline; (d) 10 to 50 mM acetate buffer, and the pH of the pharmaceutical composition is 4.6 to 5.4.

[0047] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 10 to 80 mg / mL of the anti-RANKL-NGF bispecific antibody; (b) 0.01 to 0.6 mg / mL of polysorbate 80; (c) 25 to 250 mM proline; (d) 10 to 50 mM acetate buffer, and the pH of the pharmaceutical composition is 4.6 to 5.4.

[0048] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 20 to 80 mg / mL of the anti-RANKL-NGF bispecific antibody; (b) 0.01 to 0.4 mg / mL of polysorbate 80; (c) 210-270 mM proline; (d) 10 to 30 mM acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.6 to 5.4.

[0049] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 63 to 77 mg / mL of anti-RANKL-NGF bispecific antibody; (b) 0.01 to 0.2 mg / mL polysorbate 80; (c) 210-270 mM proline; (d) 10 to 30 mM acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0050] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 63 to 77 mg / mL of anti-RANKL-NGF bispecific antibody; (b) 0.01 to 0.2 mg / mL polysorbate 80; (c) 25 to 250 mM proline; (d) 10 to 20 mM acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0051] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 63 to 77 mg / mL of anti-RANKL-NGF bispecific antibody; (b) 0.01 to 0.2 mg / mL polysorbate 80; (c) 240 mM proline; (d) 10 to 20 mM acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0052] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 63 to 77 mg / mL of anti-RANKL-NGF bispecific antibody; (b) 0.05 to 0.15 mg / mL of polysorbate 80; (c) 210-270 mM proline; (d) 16 to 24 mM acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0053] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 63 to 77 mg / mL of anti-RANKL-NGF bispecific antibody; (b) 0.08 to 0.12 mg / mL polysorbate 80; (c) 210-270 mM proline; (d) 16 to 24 mM acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0054] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) about 70 mg / mL of an anti-RANKL-NGF bispecific antibody; (b) about 0.1 mg / mL polysorbate 80; (c) about 240 mM proline; (d) about 20 mM acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0055] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 70 mg / mL of anti-RANKL-NGF bispecific antibody; (b) 0.1 mg / mL polysorbate 80; (c) 240 mM proline; (d) 20 mM acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0056] In some embodiments, the pharmaceutical composition is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0057] The present disclosure further provides a lyophilized formulation, which, after being reconstituted, can form any one of the pharmaceutical compositions described above.

[0058] The present disclosure further provides a lyophilized formulation, which is a formulation of any one of the pharmaceutical compositions described above in lyophilized form.

[0059] The present disclosure further provides a method for preparing a lyophilized formulation, comprising lyophilizing any one of the pharmaceutical compositions described above. In some embodiments, the lyophilization described in any one of the above comprises the steps of pre-freezing, primary drying, and secondary drying, in that order.

[0060] The present disclosure further provides a lyophilized preparation obtained by lyophilizing any one of the pharmaceutical compositions described above.

[0061] The present disclosure further provides a reconstituted solution, which is obtained by reconstituting any one of the above-described freeze-dried formulations.

[0062] The present disclosure further provides a reconstituted solution, which is a formulation in a reconstituted form of any one of the freeze-dried formulations described above.

[0063] In some embodiments, the reconstituted solution according to any one of the above has the same components and content as those of the pharmaceutical composition.

[0064] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 1 to 150 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 to 1.0 mg / mL of a surfactant, (c) 1 to 300 mM of a stabilizer, and (d) 5 to 100 mM of a buffer, and the pH of the pharmaceutical composition is 4.2 to 7.0.

[0065] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 1 to 150 mg / mL of any one of the anti-RANKL-NGF bispecific antibodies described above, (b) 0.01 to 1.0 mg / mL of a surfactant, (c) 1 to 300 mM of proline, and (d) 5 to 100 mM of a buffering agent, and the pH of the pharmaceutical composition is 4.2 to 7.0.

[0066] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 10 to 80 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 to 0.6 mg / mL of polysorbate 20 or polysorbate 80, (c) 25 to 290 mM of proline, and (d) 10 to 50 mM of an acetate buffer, and the pH of the pharmaceutical composition is 4.6 to 5.4.

[0067] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 10 to 80 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 to 0.6 mg / mL of polysorbate 80, (c) 25 to 250 mM of proline, and (d) 10 to 50 mM of an acetate buffer, and the pH of the pharmaceutical composition is 4.6 to 5.4.

[0068] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 20 to 80 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 to 0.4 mg / mL of polysorbate 80, (c) 210 to 270 mM of proline, and (d) 10 to 30 mM of an acetate buffer, and the pH of the pharmaceutical composition is 4.6 to 5.4.

[0069] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 63 to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.01 to 0.2 mg / mL of polysorbate 80, (c) 210 to 270 mM of proline, and (d) 10 to 30 mM of an acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0070] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 63 to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.01 to 0.2 mg / mL of polysorbate 80, (c) 25 to 250 mM of proline, and (d) 10 to 20 mM of an acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0071] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 63 to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.01 to 0.2 mg / mL of polysorbate 80, (c) 240 mM of proline, and (d) 10 to 20 mM of an acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0072] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 63 to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.05 to 0.15 mg / mL of polysorbate 80, (c) 210 to 270 mM of proline, and (d) 16 to 24 mM of an acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0073] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) 63 to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.08 to 0.12 mg / mL of polysorbate 80, (c) 210 to 270 mM of proline, and (d) 16 to 24 mM of an acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0074] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition comprises (a) approximately 70 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) approximately 0.1 mg / mL of polysorbate 80, (c) approximately 240 mM of proline, and (d) approximately 20 mM of an acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0075] In some embodiments, the reconstitution solution of any one of the above is a solution containing the following components: The pharmaceutical composition contains (a) 70 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.1 mg / mL of polysorbate 80, (c) 240 mM of proline, and (d) 20 mM of an acetic acid-sodium acetate buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0076] In some embodiments, the pharmaceutical composition or reconstituted solution described above is a subcutaneous injection formulation, an intravenous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation. In some embodiments, the pharmaceutical composition or reconstituted solution described above is a subcutaneous injection formulation.

[0077] In some embodiments, the pharmaceutical composition or reconstituted solution described above is suitable for subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection, and preferably suitable for subcutaneous injection.

[0078] In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation described above is used to prepare a drug for subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection, preferably, it is used to prepare a drug for subcutaneous injection.

[0079] The present disclosure further provides a kit comprising at least one container, each container independently containing any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, or any one of the reconstitution solutions described above.

[0080] In some embodiments, the present disclosure further provides a method for diagnosing, treating, or alleviating a condition in a subject, comprising administering to the subject an effective amount of any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above.

[0081] In some embodiments, the present disclosure further provides use of any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above in the preparation of a medicament for treating or preventing a disease.

[0082] In some embodiments, the present disclosure further provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above.

[0083] In some embodiments, the present disclosure further provides any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above, which are used for treating or preventing a disease.

[0084] In one aspect, the present disclosure further provides use of any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above in the preparation of a medicament for preventing or treating a disease or condition.

[0085] In some embodiments, the disease described in any one of the above is pain, joint stiffness, or bone loss.

[0086] In some embodiments, the pain is selected from osteoarthritic pain, rheumatoid arthritis pain, gout, bone cancer pain, fracture pain, post-operative pain, cancer pain, bladder pain syndrome, musculoskeletal pain, prostatitis (e.g., chronic prostatitis), pelvic pain (e.g., chronic pelvic pain), interstitial cystitis, lower back pain, dysmenorrhea, pain associated with bone disease, trigeminal neuralgia, post-herpetic neuralgia, herpes zoster infection, sciatica, migraine, diabetic neuropathy, and peripheral nerve-related pain.

[0087] In some embodiments, the bone loss is associated with at least one condition selected from the group consisting of osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, bone loss, osteoporosis, osteonecrosis, bone injury, bone resorption, osteogenesis imperfecta, inflammation, autoimmune disease, enteritis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, periodontal bone resorption, osteolytic metastasis, and cancer.

[0088] In some embodiments, the cancer is selected from breast cancer, prostate cancer, thyroid cancer, renal cancer, lung cancer, esophageal cancer, rectal cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, melanoma, multiple myeloma, osteosarcoma, lymphoma, non-small cell lung cancer, bone tumors, and Hodgkin's disease.

[0089] In some embodiments, the disease is an NGF or RANKL-associated disease.

[0090] In some embodiments, the disease is an NGF or RANKL-expressing disease.

[0091] In one aspect, the present disclosure provides a method for treating or preventing a disease associated with NGF or RANKL, the method comprising administering to a subject a prophylactically or therapeutically effective amount of any one of the pharmaceutical compositions described above, the lyophilized formulation described above, the reconstituted solution described above, or the kit described above.

[0092] In some embodiments, the NGF- or RANKL-associated disorder is pain, joint stiffness, or bone loss.

[0093] In one aspect, any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above of the present disclosure can be used as a medicament. In some embodiments, it is used as a medicament for treating pain, joint stiffness, or bone loss. In some embodiments, it is used as a medicament for treating osteoarthritis pain, rheumatoid arthritis pain, gout, bone cancer pain, fracture pain, postoperative pain, cancer pain, bladder pain syndrome, musculoskeletal pain, prostatitis (e.g., chronic prostatitis), pelvic pain (e.g., chronic pelvic pain), interstitial cystitis, lower back pain, dysmenorrhea, bone disease-related pain, trigeminal neuralgia, postherpetic neuralgia, herpes zoster infection, sciatica, migraine, diabetic neuropathy, and peripheral nerve-related pain. In some embodiments, it is used as a pharmaceutical agent to treat osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, bone loss, osteoporosis, osteonecrosis, bone damage, bone resorption, osteogenesis imperfecta, inflammation, autoimmune diseases, enteritis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, periodontal bone resorption, osteolytic metastasis, and bone loss due to cancer.

[0094] In one aspect, the present disclosure provides use of any one of the pharmaceutical compositions, any one of the lyophilized formulations, any one of the reconstituted solutions, or any one of the kits in the preparation of a medicament for preventing or treating a disease associated with NGF or RANKL. In some embodiments, the disease associated with NGF or RANKL is pain, joint stiffness, or bone loss.

[0095] In one aspect, any one of the pharmaceutical compositions described above, any one of the lyophilized preparations described above, any one of the reconstituted solutions described above, or any one of the kits described above provided in the present disclosure can be used as a medicament for preventing or treating a disease associated with NGF or RANKL. In some embodiments, the disease associated with NGF or RANKL is pain, joint stiffness, or bone loss. [Brief explanation of the drawings]

[0096] [Figure 1]1 is a schematic diagram of the structure of DVD-IgG. [Figure 2A] Figure 2A shows the statistical results of mouse pain behavior on day 14 of combination administration, Figure 2B shows the statistical results of mouse pain behavior on day 21 of combination administration, Figure 2C shows the scoring results of mouse bone damage on day 14 of combination administration, and Figure 2D shows the scoring results of mouse bone damage on day 21 of combination administration, where **** vs. vehicle, P<0.0001, *** vs. vehicle, P<0.001, ** vs. vehicle, P<0.01, * vs. vehicle, P<0.05. Blank control (Blank), sham-operated group (Sham), vehicle control (vehicle). [Figure 2B] Same as above [Figure 2C] Same as above [Figure 2D] Same as above [Figure 3] Figure 3A shows the statistical results of pain behavior in mice treated with bispecific antibody 1 on day 14, and Figure 3B shows the statistical results of pain behavior in mice treated with bispecific antibody 1 on day 21, where **** vs vehicle, P<0.0001, *** vs vehicle, P<0.001, ** vs vehicle, P<0.01, and * vs vehicle, P<0.05, respectively. [Figure 4-1] Figure 4A shows the statistical results of pain behavior in mice treated with bispecific antibody 1 on day 15. Figure 4B shows the statistical results of pain behavior in mice treated with bispecific antibody 1 on day 21. Figure 4C shows the scoring results of bone damage in mice treated with bispecific antibody 1 on day 15. Figure 4D shows the scoring results of bone damage in mice treated with bispecific antibody 1 on day 21. where, **** vs. vehicle, P<0.0001; *** vs. vehicle, P<0.001; ** vs. vehicle, P<0.01; * vs. vehicle, P<0.05. Sham-operated group (Sham), vehicle control (vehicle). [Figure 4-2] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0097] term In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0098] As used in this disclosure, the singular forms "a," "an," and "the above" include plural referents unless the context clearly dictates otherwise.

[0099] Unless the context clearly indicates otherwise, in the patent specification and claims, the words "comprises," "has," "includes," and the like are to be understood as meaning "including, but not limited to," rather than in an exclusive or exhaustive sense.

[0100] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs or does not occur.

[0101] Those skilled in the art should understand that when a reference range, cutoff value, or specific value is used, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of difference of up to 20% (i.e., ±20%). Because many numerical values ​​used herein are determined experimentally, those skilled in the art should understand that such determinations can and usually do vary between different experiments. Due to such inherent variations, the values ​​used herein should not be unduly limited. Thus, the term "about" is used to include a variation of ±20% or less, a variation of ±10% or less, a variation of ±5% or less, a variation of ±1% or less, a variation of ±0.5% or less, or a variation of ±0.1% or less from a particular value.

[0102] Although the present disclosure provides content ranges or content values, it will be understood by those skilled in the art that the content ranges or content values ​​cover an acceptable margin of error for the specific values ​​measured.

[0103] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0104] "Anti-RANKL antibody" refers to an antibody that binds to RANKL or an epitope thereof and is capable of inhibiting the biological activity of RANKL and / or inhibiting the downstream pathway of RANKL.

[0105] The term "NGF," nerve growth factor, refers to nerve growth factor and variants thereof that retain at least some of the biological activity of NGF. As used herein, NGF includes wild-type sequence NGF or naturally occurring variants thereof of any mammalian species, including human, murine, simian, canine, feline, equine, or bovine.

[0106] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon bonded to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that act in a manner similar to a naturally occurring amino acid.

[0107] The term "antibody" is used in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments or portions thereof), as long as they exhibit the desired antigen-binding activity. A "native antibody" is a naturally occurring immunoglobulin molecule. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain contains one variable region (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains one variable region (VL), also called a variable light chain domain or light chain variable domain, followed by one constant light chain domain (light chain constant region, CL). The term "bispecific antibody" refers to an antibody (including antibodies or antigen-binding fragments thereof, e.g., single chain antibodies) that can specifically bind to two different antigens or at least two different antigenic epitopes on the same antigen.

[0108] The term "variable region" or "variable domain" refers to the domain in an antibody's heavy or light chain that is involved in binding to an antibody antigen. As used herein, the heavy chain variable region (VH) and light chain variable region (VL) of an antibody each contain four conserved framework regions (FR) and three complementarity-determining regions (CDR). The term "complementarity-determining region" or "CDR" refers to the region in the variable region that primarily promotes antigen binding, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, while VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL is sufficient to confer antigen-binding specificity.

[0109] The amino acid sequence boundaries of CDRs can be determined by various known methods, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16;9:2278), and the correspondence between various numbering systems is well known to those of skill in the art. The numbering conventions of the present disclosure are as shown in Table 1 below.

[0110] [Table 1]

[0111] Unless otherwise specified, the variable region and CDR sequences in the examples of the present disclosure are all based on the "Kabat" numbering system. In specific embodiments, the Kabat numbering system is used to define amino acid residues, but technical solutions corresponding to other numbering systems are considered equivalent technical solutions.

[0112] A "pharmaceutical composition" is meant to contain one or more bispecific antibodies described herein and other ingredients, such as physiological / medicinal carriers and excipients. The pharmaceutical composition is intended to facilitate administration to the body and contribute to the absorption of the active ingredients to further exert their biological activity. In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0113] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to allow or facilitate diagnosis. The effective amount used for a particular subject or veterinary subject can vary depending on factors such as the condition being treated, the subject's overall health, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0114] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are prepared by well-known conventional methods.

[0115] "Buffer" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0116] A "histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, and histidine-histidine sulfate, with histidine-histidine hydrochloride being preferred. Histidine-histidine hydrochloride buffers may be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

[0117] A "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. A preferred citrate buffer is citric acid-sodium citrate.

[0118] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium salt, succinic acid-potassium succinate, succinic acid-calcium salt, etc. A preferred succinate buffer is succinic acid-sodium salt. Illustratively, the succinic acid-sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate salt.

[0119] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include citric acid-disodium hydrogen phosphate, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, etc. A preferred phosphate buffer is citric acid-disodium hydrogen phosphate.

[0120] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate.

[0121] A "poloxamer" is a block copolymer of oxirane and epoxypropane that is water-soluble and is used as a surfactant in drug formulations. Examples of poloxamers include poloxamer 188.

[0122] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a freeze-drying step is performed on a liquid or solution formulation.

[0123] The pharmaceutical compositions described herein can achieve stable efficacy, i.e., the antibodies therein essentially retain their physical and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions essentially retain their physical and chemical stability and biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are several analytical techniques for measuring protein stability, which can be used to measure stability after storage at a predetermined temperature for a predetermined period of time.

[0124] A stable formulation is one that shows no significant change when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years. Stable liquid formulations also include liquid formulations that exhibit desired characteristics after storage at temperatures including 25°C for periods including 2 weeks, 4 weeks, 1 month, 3 months, or 6 months. Stable liquid formulations further include liquid formulations that exhibit desired characteristics after storage at temperatures including 40°C for periods including 2 weeks, 4 weeks, 1 month, 3 months, or 6 months. A typical example of stability is determined by SEC-HPLC, where the amount of antibody that has undergone aggregation or degradation typically does not exceed about 10%, preferably does not exceed about 5%. Visual analysis reveals that the formulation is a pale yellow, almost colorless, or colorless liquid, or is clear to slightly opalescent. The concentration, pH, and osmolality of the formulation do not vary by more than ±10%, preferably by more than ±5%. The formulations typically form no more than about 10%, preferably no more than about 5% aggregates.

[0125] An antibody "retains its physical stability" in a drug formulation if it does not exhibit significant increased aggregation, precipitation, and / or denaturation as determined by visual inspection of color and / or clarity or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines tertiary protein structure) and by FTIR spectroscopy (which determines secondary protein structure).

[0126] An antibody "retains its chemical stability" in a drug formulation if it does not undergo significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or cleavage (e.g., assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (e.g., assessed by methods such as peptide mapping coupled with mass spectrometry or MALDI / TOF / MS), deamidation (e.g., assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, or isoaspartic acid content measurement), and isomerization (e.g., assessed by isoaspartic acid content measurement, peptide mapping, etc.).

[0127] An antibody "retains its biological activity" in a drug formulation if the biological activity of the antibody over a given period of time is within a given range of biological activity exhibited at the time the drug formulation was prepared.

[0128] "Administration," "giving," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Administration," "giving," and "treatment" also refer to ex vivo and in vitro treatment, e.g., of cells, with a reagent, diagnostic, binding composition, or through another cell. "Treatment," when applied to a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0129] "Treatment" refers to providing an internal or external therapeutic agent, including, for example, any one of the pharmaceutical compositions disclosed herein, to a patient having one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is provided with a therapeutic agent in an amount that effectively alleviates one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting those symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or product) may be ineffective in alleviating each target disease symptom, but should reduce the target disease symptom in a statistically significant number of patients, as determined by any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0130] The pharmaceutical compositions of the present disclosure can be administered by any suitable means, including parenteral, pulmonary, and intranasal administration, and, when localized treatment is required, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion. In some embodiments, the pharmaceutical compositions of the present disclosure are administered by subcutaneous injection.

[0131] The pharmaceutical compositions of the present disclosure are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific disease being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disease, the delivery site of the reagent, the method of administration, the administration schedule, and other factors known to medical professionals. Optionally, the pharmaceutical composition may be further formulated with one or more other reagents used in the prevention or treatment of the disease. The effective amount of such other reagents will depend on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of disease or treatment, and other factors. They may be used in the same dosages and via any route of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically confirmed to be appropriate.

[0132] In the present disclosure, the disease associated with NGF or RANKL is not limited as long as it is an NGF- or RANKL-related disease. For example, the therapeutic response induced by the antibody of the present disclosure can bind to human NGF or RANKL, thereby inhibiting the binding of NGF or RANKL to its receptor, or killing cells that overexpress NGF or RANKL, or inhibiting the proliferation of cells that overexpress NGF or RANKL.

[0133] The above specification provides details of one or more embodiments of the present disclosure. Although the present disclosure can be practiced or tested using any methods and materials similar or equivalent to those described herein, the preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate preferred embodiments of the present disclosure. These examples should not be construed in any way as limiting the scope of the present disclosure, which is limited only by the claims.

[0134] Example - Preparation and detection of anti-RANKL-NGF bispecific antibodies PCT / CN2022 / 092333 (filing date: 2022.05.12, priority patent application number: CN202110515444.9) is incorporated herein by reference in its entirety.

[0135] Example 1: Preparation of anti-RANKL antibodies A yeast library was constructed by designing primers. The library was enriched and screened using RANKL protein (Sino biological, 11682-HNCH) to obtain candidate clones, and the amino acid sequences of the antibody light and heavy chain variable regions in the clones were determined by sequencing. The sequences of the obtained exemplary anti-RANKL antibodies are as follows:

[0136] [Table 2]

[0137] [Table 3]

[0138] The variable regions were fused to human light and heavy chain constant regions to form the light and heavy chains of a complete antibody. Exemplary antibody constant region sequences are as follows:

[0139] human IgG4 heavy chain constant region, [ka] SEQ ID NO: 9, human kappa light chain constant region, [ka] Sequence number 10.

[0140] The sequences of exemplary anti-RANKL antibodies are as follows:

[0141] D75H heavy chain, [ka] SEQ ID NO: 11, D75H light chain, [ka] Sequence number 12.

[0142] The sequence of the control anti-RANKL antibody is as follows:

[0143] Denosumab heavy chain sequence, [ka] SEQ ID NO: 13, Denosumab light chain sequence, [ka] Sequence number 14.

[0144] Example 2: Preparation of anti-RANKL / NGF bispecific antibodies The second antigen-binding domain that binds to NGF of the bispecific antibodies of the present disclosure may be derived from the antigen-binding portion of any suitable antibody. Particularly suitable antibodies are described, for example, in International Application WO2004058184A2 (incorporated herein by reference in its entirety).

[0145] The CDR and variable region sequences of the NGF binding domain of an exemplary bispecific antibody are as follows:

[0146] [Table 4]

[0147] NGF binding domain heavy chain variable region, [ka] SEQ ID NO: 21, NGF binding domain light chain variable region, [ka] Sequence number 22.

[0148] The variable region of the NGF binding domain was fused to the heavy chain constant region of SEQ ID NO: 9 or SEQ ID NO: 23 and the light chain constant region of SEQ ID NO: 10, respectively, to obtain an anti-NGF antibody. The sequences are as follows:

[0149] [ka] SEQ ID NO: 23, anti-NGF antibody (N-mAb) heavy chain, [ka] SEQ ID NO: 24, anti-NGF antibody (N-mAb) light chain, [ka] SEQ ID NO: 25, Sequence of control anti-NGF antibody, Tanezumab heavy chain, [ka] SEQ ID NO: 26, Tanezumab light chain, [ka] Sequence number 27.

[0150] Currently, clinical dosages of monoclonal antibodies targeting NGF and RANKL for the indication of bone metastasis vary widely. Therefore, when preparing bispecific antibodies against NGF and RANKL, the activities of the NGF and RANKL arms of the bispecific antibody must be balanced to determine the appropriate dosage, so as to avoid side effects due to excessive dosing or failure of the bispecific antibody to function due to excessive dosing.

[0151] The bispecific antibody prepared according to the present disclosure has a DVD-IgG structure (shown in Figure 1). The light chain variable domains (VL) of an anti-RANKL antibody and an anti-NGF antibody are tandemly linked, either directly or via a short linker using recombinant DNA technology, followed by a light chain constant domain. Similarly, the heavy chain contains two tandemly linked heavy chain variable domains (VH), followed by a constant domain CH1 and an Fc region. Examples of linkers include, but are not limited to, peptide linkers such as ASTKGP (SEQ ID NO: 28) and TVAAP (SEQ ID NO: 29).

[0152] The sequence of an exemplary bispecific antibody is as follows:

[0153] First chain of bispecific antibody 1 [ka] SEQ ID NO: 30, Second chain of bispecific antibody 1 [ka] Sequence number 31.

[0154] Note: In the sequences, the single underlined parts are the RANKL antibody variable regions, the double underlined parts are the NGF antibody variable regions, the bold parts are linkers, and the remaining sequences are constant regions.

[0155] Test Example Test Example 1. Affinity test of anti-RANKL antibodies A fixed amount of antibody to be measured was affinity-captured using a biosensor chip, Protein A (GE, 29127556). A gradient series of antigens, human RANKL protein (Sino Biological, 11682-HNCH) and human NGF protein (Sino Biological, 11050-HNAC), was then passed over the chip surface. The reaction signals were detected in real time using a Biacore™ system to obtain binding and dissociation curves. After each dissociation cycle, the biochip was regenerated by washing with a pH 1.5 glycine-HCl regeneration solution (GE, BR-1003-54). The experimental data were fitted with a 1:1 model using BIAevaluation version 4.1 software to obtain affinity values. The results are shown in Table 5 below.

[0156] [Table 5]

[0157] The results showed that the affinity of the modified antibody D75H was more than six times higher than that of the Denosumab antibody.

[0158] Test Example 2: Detection of the ability of anti-RANKL antibodies to inhibit the binding of RANKL to RANK The inhibitory activity of antibodies against ligand-receptor binding was detected by ELISA. Human RANK protein was diluted to 2 μg / mL in PBS (Gensei B320) buffer, pH 7.4, and added to a 96-well microplate (Corning, 3590) at a volume of 100 μL per well. The plate was then incubated overnight at 4°C. After discarding the liquid, 200 μL of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking and incubation at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (PBS, pH 7.4, containing 0.1% Tween-20) before use. A fixed concentration of biotin-labeled human RANKL protein (Sino Biological, 11682-HNCH) was mixed with gradient-diluted antibody and pre-incubated at 37°C for 30 minutes. The mixture was then added to a blocked microplate and incubated at 37°C for 1.5 hours. After incubation, the plate was washed three times with PBST, and 100 μL of streptavidin-HRP (Invitrogen, 434323, diluted 1:4000) was added to each well and incubated at 37°C for 1 hour. The supernatant was discarded, and the plate was washed three times with PBST. Then, 100 μL of TMB chromogenic substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes. The reaction was stopped by adding 450 μL of 1 M H2SO4 to each well, and the absorbance at 450 nm was read using a plate reader. The ligand-receptor binding inhibition curve was fitted using software to determine the IC 50 The values ​​were calculated, and the results are shown in Table 6 below.

[0159] [Table 6]

[0160] The results showed that the anti-RANKL antibody D75H was able to inhibit the binding of RANKL to RANK, and its inhibitory activity was superior to that of the denosumab antibody.

[0161] Test Example 3. Detection of binding activity of bispecific antibodies to antigens of different species by ELISA The binding activity of the bispecific antibodies of the present disclosure to human RANKL (Sino biological, 11682-HNCH), monkey RANKL (Sino Biological, 90301-C01H), mouse RANKL (R&D Systems, 462-TR / CF), human NGF (the monkey NGF sequence is identical to that of human NGF) (Sino biological, 11050-HNAC), and mouse NGF (Sino Biological, 50385-MNAC) was detected by ELISA, as follows:

[0162] Antigens were diluted to 1 μg / mL in pH 7.4 PBS buffer (Gensei Biosciences, B320) and added to a 96-well microplate (Corning, 9018) at a volume of 100 μL per well and incubated at 4°C overnight. After discarding the liquid, 300 μL per well of a blocking solution of 5% nonfat milk (BD, 232100) diluted in PBS was added and incubated at 37°C for 2 hours. After discarding the blocking solution, the plate was washed three times with PBST buffer (PBS, pH 7.4, 0.1% Tween-20). Then, 100 μL of bispecific antibodies at different concentrations diluted in sample diluent (PBS, pH 7.4, 1% BSA) was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST, and 100 μL of HRP-labeled anti-human Fc secondary antibody (Abcam, ab97225) diluted with sample diluent was added to each well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB chromogenic substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes. The reaction was stopped by adding 50 μL of 1M H2SO4 to each well, and the absorbance value at 450 nm was read using a plate reader to determine the EC of the bispecific antibody binding to the antigen. 50 The values ​​were calculated and the results are shown in Table 7 below.

[0163] [Table 7]

[0164] The results showed that the binding activity of Bispecific Antibody 1 to both human RANKL and monkey RANKL was superior to that of the control antibody Denosumab. However, neither Bispecific Antibody 1 nor Denosumab cross-linked mouse RANKL. The binding activity of Bispecific Antibody 1 to both human NGF and mouse NGF was clearly weaker than that of Tanezumab, indicating that Bispecific Antibody 1 of the present disclosure successfully reduced the binding activity of the Tanezumab terminus (the second antigen-binding domain that specifically binds to NGF).

[0165] Test Example 4. Affinity test of bispecific antibodies A fixed amount of antibody to be measured was affinity-captured using a biosensor chip, Protein A (GE, 29127556). A gradient series of antigens, human RANKL protein (Sino Biological, 11682-HNCH) and human NGF protein (Sino Biological, 11050-HNAC), was then passed over the chip surface. The reaction signals were detected in real time using a Biacore™ system to obtain binding and dissociation curves. After each dissociation cycle, the biochip was regenerated by washing with a pH 1.5 glycine-HCl regeneration solution (GE, BR-1003-54). The experimental data were fitted with a 1:1 model using BIAevaluation version 4.1 software to obtain affinity values. The results are shown in Table 8 below.

[0166] [Table 8]

[0167] The results showed that the affinity of Bispecific Antibody 1 for human RANKL was 3.8-fold higher than that of Denosumab, and the affinity of Bispecific Antibody 1 for human NGF was significantly lower than that of Tanezumab.

[0168] Test Example 5: Ligand and receptor inhibition experiment The inhibitory activity of antibodies against the ligand and receptor was detected using ELISA. Receptor proteins (RANK or TrkA) were diluted to 2 μg / mL in pH 7.4 PBS (Genbioso Biosciences, B320) buffer, added to a 96-well microplate (Corning, 3590) at a volume of 100 μL per well, and incubated overnight at 4°C. After discarding the liquid, 200 μL of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking and incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (PBS, pH 7.4, containing 0.1% Tween-20) before use. A fixed concentration of biotin-labeled ligand protein (RANKL or NGF) was mixed with gradient-diluted antibody or fusion protein and preincubated at 37°C for 30 minutes. The mixture was then added to a blocked microplate and incubated at 37°C for 1.5 hours. After incubation, the plate was washed three times with PBST. 100 μL of streptavidin-HRP (Invitrogen, 434323, diluted 1:4000) was added to each well and incubated at 37°C for 1 hour. The supernatant was discarded, and the plate was washed three times with PBST. 100 μL of TMB chromogenic substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes. The reaction was stopped by adding 50 μL of 1 M H2SO4 to each well. The absorbance at 450 nm was measured using a plate reader, and the IC2000 curve was fitted using software to calculate the inhibition of ligand-receptor binding. 50 The value was calculated.

[0169] Information on the ligand and receptor proteins used in the experiments includes human RANKL protein (Sino Biological, 11682-HNCH), human RANK protein (Sino Biological, 16078-H02H), human TrkA protein (Sino Biological, 11073-H03H), and human NGF protein (Sino Biological, 11050-HNAC).

[0170] The results of the experiment are shown in Table 9 below.

[0171] [Table 9]

[0172] The results showed that bispecific antibody 1 had superior inhibitory activity against RANKL to denosumab and significantly weaker inhibitory activity against NGF than tanezumab, indicating that reducing the binding activity of the NGF terminus of bispecific antibody 1 significantly reduced its inhibitory activity against NGF.

[0173] Test Example 6: Osteoclast differentiation experiment Since the main function of RANKL is to promote the differentiation and maturation of osteoclasts, the inhibitory activity of antibodies against RANKL can be tested by osteoclast differentiation experiments. The experimental method is as follows.

[0174] Raw264.7 cells (ECACC, 91062702) were plated in a 96-well cell culture plate (Corning, 3599) and cultured overnight at 37°C. The next day, a fixed concentration of human RANKL protein (Sino Biological, 11682-HNCH) and gradient dilutions of the bispecific antibody to be assayed were added to the 96-well cell plate and cultured at 37°C for 4 days. The 96-well plate was removed, the supernatant was removed, and 100 μL of cell lysis solution (Bidomtian, P0013J) was added to each well. After mixing thoroughly by pipetting, the cell lysate was transferred to a centrifuge tube, incubated on ice for 10 minutes, and centrifuged to remove the supernatant. Refer to the method in the Tartrate-Resistant Acid Phosphatase Detection Reagent Kit (Hekidontian, P0332), take 40 μL of sample to be detected, add 40 μL of colorimetric substrate and 5 μL of tartaric acid, mix thoroughly, and incubate in a 37°C incubator for 10 min. Add 160 μL of stop solution, measure the OD405nm value with a plate reader, and calculate the IC by fitting the data with software. 50 The experimental results are shown in Table 10 below.

[0175] [Table 10]

[0176] The results showed that Bispecific Antibody 1 had 3.3 times more inhibitory activity against osteoclast differentiation than Denosumab.

[0177] Test Example 7. TF-1 cell proliferation test Since NGF can promote the proliferation of TF-1 cells in vitro, the inhibitory activity of the antibodies against NGF was evaluated in a TF-1 cell proliferation experiment. The experimental method is as follows.

[0178] TF-1 cells (ATCC, CRL-2003) were resuspended in 1640 medium (Gibco, 22400-105) without GM-CSF and plated onto a 96-well cell culture plate (Corning, 3903) and cultured overnight at 37°C. The next day, a fixed concentration of human NGF protein (Sino biological, 11050-HNAC) and gradient dilutions of the antibody to be assayed were added to the 96-well plate and cultured for 72 hours at 37°C. The plate was removed, and 50 μL of Cell-titer Glo (Promega, G755B) solution was added to each well. The plate was gently shaken for 10 minutes to ensure uniform mixing, then incubated at room temperature for 10 minutes. Bioluminescence signals were detected using a PE Victor 3. Data were fitted using software to calculate IC values. 50 The experimental results are shown in Table 11 below.

[0179] [Table 11]

[0180] The results showed that bispecific antibody 1 could still inhibit TF-1 cell proliferation, and the inhibitory activity of tanezumab was approximately 4.2-fold higher than that of bispecific antibody 1, indicating that bispecific antibody 1 significantly reduced the activity of NGF-terminal.

[0181] Therefore, assuming that in vitro activity correlates with dosage, it is predicted that the dosage of RANKL terminus can be reduced to about 40 mg while still maintaining the in vivo efficacy of RANKL terminus, and the dosage of NGF terminus can be increased to about 80 mg while still maintaining good safety. Furthermore, since the administration cycle of the anti-NGF antibody Tanezumab is currently twice that of the anti-RANKL antibody Denosumab in clinical practice, the exemplary bispecific antibody 1 of the present disclosure can balance the dosages of the two targets.

[0182] Test Example 8. In vivo efficacy of bone metastasis animal model The analgesic and osteoprotective effects of the antibody were evaluated using a bone metastasis animal model.

[0183] To construct the model, male C57 BL / 6 mice (SPF grade) were purchased from Changzhou Cavest Co., Ltd. They were housed in a laboratory environment with a 12 / 12-hour light / dark cycle, a temperature of 23°C ± 1°C, and humidity of 40-50%. All animals were fed standard sterile mouse chow and had free access to water and food. Model construction began when the mice reached a weight of approximately 25g.

[0184] Mice were anesthetized with an intraperitoneal injection of 1% sodium barbital and then skin preparation was performed. The mice were placed on a heat pad and a 1 cm incision was made with ophthalmic scissors on the lateral skin of the left hind knee parallel to the femur, exposing the muscle. The skin and muscle layer were then bluntly separated. Using the connective tissue line as a guide, an incision was made between the rectus femoris and vastus medialis muscles with ophthalmic scissors. The rectus femoris and patella were then moved medially with tweezers to expose the femoral condyle without cutting the patellar ligament. A 0.45 mm diameter needle was used to drill a hole in the ankle fossa from the top to the center of the femur, and the needle was inserted 1-1.5 cm into the intramedullary cavity to create an injection route. A Hamilton microsyringe (50 μL, Model 1705 RN SYR, No. 26 needle, Model ga26 / 51mm / pst3, Product No. 7768-02) was inserted into the intrathecal cavity through the opening, and 10 μL (5 × 10) of LLC1 cells (ATCC, CRL-1642) was added. 4 ) was slowly injected. After the injection was completed, the needle was removed, the leg was straightened, the patella and ligaments were repositioned with tweezers, the muscles were repositioned, antibiotic powder was sprinkled, and the incision was sutured with an automatic suture clip (Roboz, Reflex 7 Clip 7mm). After surgery, animals were housed in cages of no more than three animals, and the suture clips were removed after seven days. The sham-operated group was operated on as described above, but the same volume of PBS was injected into the intrathecal cavity. The blank group was maintained in a normal environment without any treatment. Administration began 7 days after model creation, and was administered once every five days for a total of three doses. The mice's pain behavior and femoral damage were evaluated on days 14 (or 15) and 21, respectively.

[0185] For statistical analysis of pain behavior, the animals were placed on a floor surrounded by organic glass and covered with a wire grid. They were allowed to adapt for 30 minutes (until the animals stopped exploring the box and grooming). Their movements were then observed, and any behaviors that attempted to avoid the pain in the affected limb within 5 minutes were evaluated. The duration of these behaviors was measured using a stopwatch.

[0186] Pain avoidance behavior is defined as follows:

[0187] (1) Complete protection (elevating the affected limb to avoid bearing the weight of the landing when walking), (2) Contracting the five fingers of the affected limb and breaking through the grid (a normal mouse's limbs should have the five fingers extended and spread flat on the grid, supported by the force of the front soles of the feet. In tumor-bearing bone metastasis mice, the affected limb will have the five fingers contracted. In this case, if the mouse can land on its hind heel and support itself, the pain is considered relatively mild and is not counted in the measurement time. However, if the entire sole of the foot does not come into contact with the grid or breaks through the grid, it is counted in the measurement time). (3) Licking the affected limb (4) sporadic one-legged jumping, and (5) Standing on one hind leg (both forelimbs raised).

[0188] How bone damage is scored; The left femur was carefully stripped in the prone position, and bone destruction was detected using an MX-20 digital cabinet X-ray system (Faxitron / Bioptics), and bone damage was scored by a single fixation expert.

[0189] The scoring criteria are as follows:

[0190] 0 - relatively normal, 1 - mild - focal damage, 2 - moderate - focal damage, 3 - moderate - multiple damage, 4 - severe - diffuse damage. The distal femur and the proximal femur were scored separately and then added together, with 4 + 4 = 8 points when bone destruction was at its worst.

[0191] 1. Testing the effects of RANLK antagonists and NGF antagonists on pain relief and bone protection Because denosumab does not have cross-binding activity against mouse RANKL, we used the mouse RANKL antibody AMR2 in combination with tanezumab to evaluate whether the in vivo efficacy of the combination of the two antibodies is superior to that of a single antibody.

[0192] The variable region sequences of the AMR2 antibody were derived from WO2013176469A1, and the heavy and light chain sequences of the variable regions were fused to human IgG4 and human lambda constant regions, respectively, to construct the full-length antibody AMR2. The relevant sequences for AMR2 are as follows:

[0193] [Table 12]

[0194] AMR2 heavy chain variable region, [ka] SEQ ID NO: 38, AMR2 light chain variable region, [ka] SEQ ID NO: 39, AMR2 heavy chain [ka] SEQ ID NO: 40, AMR2 light chain [ka] Sequence number 41.

[0195] The groupings and dosages are as shown in Table 13 below.

[0196] [Table 13]

[0197] NOTE: Q5d*3 indicates administration once every 5 days, a total of 3 times, and ip indicates intraperitoneal injection administration.

[0198] The experimental results are shown in the following Figures 2A to 2D.

[0199] Statistical results of the pain behavior of mice showed that on day 14, all three treatment groups showed significant analgesic effects compared to the negative group, with the combined treatment group showing a stronger analgesic effect than the monoclonal antibody group. On day 21, only the combined treatment group showed significant analgesic effects, and the two monoclonal antibody groups also showed some analgesic effects, but showed no statistical difference compared to the negative group (vs. vehicle: *P<0.05, **P<0.01, ***P<0.0001).

[0200] According to the bone damage scoring results, the combination administration group showed a certain bone protective effect on day 14. On day 21, the combination administration group showed a significant bone protective effect. Taking these experimental results together, it is clear that the combination administration group exhibited beneficial effects in both analgesia and bone protection. Therefore, the inventors then further verified the in vivo beneficial effects of the bispecific antibody molecule of the present disclosure.

[0201] 2. Evaluation of the in vivo efficacy of NGF-terminal bispecific antibodies Because the in vitro activity of the NGF terminus of bispecific antibody 1 was approximately four-fold lower than that of the NGF monoclonal antibody tanezumab, this efficacy test was conducted to confirm whether the NGF terminus of bispecific antibody 1 still had efficacy in mice. The experimental groups are shown in the table below.

[0202] [Table 14]

[0203] NOTE: Q5d stands for administration once every 5 days, and ip stands for intraperitoneal injection administration.

[0204] The experimental results are shown in FIGS. 3A and 3B.

[0205] The results showed that on days 14 and 21 after administration, bispecific antibody 1 exhibited significant analgesic effects compared to the negative group, indicating that the NGF terminus of bispecific antibody 1 still possesses significant pharmacological efficacy.

[0206] 3. Evaluation of the in vivo efficacy of bispecific antibodies A bone metastasis animal model was established using human RANKL transgenic mice (purchased from Biocytogen) to evaluate the in vivo efficacy of the bispecific antibody. The experimental groups are shown in Table 15 below.

[0207] [Table 15]

[0208] NOTE: Q5d stands for administration once every 5 days, and ip stands for intraperitoneal injection administration.

[0209] The experimental results are shown in FIGS. 4A to 4D.

[0210] The results showed that bispecific antibody 1 exhibited significant analgesic and bone-protective effects. On days 15 and 21, both the high-dose group of bispecific antibody 1 exhibited significant analgesic effects, and both the high- and low-dose groups showed significant protective effects against bone damage.

[0211] Test Example 9: PK test in rats An in vivo pharmacokinetic study was conducted using SD rats. Male SD rats (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were divided into groups of four and intravenously administered 4 mg / kg of bispecific antibody 1. From the treatment group, 0.2 mL of whole blood was collected before and 5 minutes, 8 hours, 24 hours, 48 ​​hours, 84 hours, 9 days, 10 days, 14 days, 21 days, and 28 days after administration. No anticoagulant was added. After collection, the blood was incubated at 4°C for 30 minutes and then centrifuged at 1000 g for 15 minutes. The supernatant serum was collected, placed in EP tubes, and stored at -80°C.

[0212] The serum blood concentration was detected by ELISA, and the pharmacokinetic parameters of the antibody waiting to be measured were calculated using Winnolin software. The detection results are as follows:

[0213] [Table 16]

[0214] The results showed that Bispecific Antibody 1 showed good PK results in rats, with relatively similar half-lives at both the RANKL and NGF ends, at 15.09 days and 14.3 days, respectively.

[0215] Preparation Example - Anti-RANKL-NGF Bispecific Antibody Formulation Exemplary antibody pharmaceutical composition (formulation) preparation process Step 1: A bulk formulation containing the RANKL-NGF bispecific antibody and a stabilizer was prepared, and the solution was sterilized and filtered using a 0.22 μm filter cartridge, and the filtrate was collected.

[0216] Step 2: The amount of filling was adjusted (target volume was 1.0 mL / bottle), and the vials were used for filling. Sampling was carried out at the start, during, and end of filling to detect any difference in the amount of filling.

[0217] Step 3: The capping machine was turned on and the aluminum cap was applied and crimped on.

[0218] Step 4: Visually inspect the product to see if there were any defects such as incorrect filling amount, poor appearance, etc. Printed carton labels, folded the cartons, placed them in cartons, and attached the carton labels.

[0219] exterior The sample bottle was wiped clean, and the color, transparency, and visible foreign matter of the sample were visually observed under a white background and a black background of a transparency meter at an illumination intensity of 1000 to 1500 lx, respectively.

[0220] Appearance inspection machine: Seitakuki YB-2A transparency measuring instrument.

[0221] Melting temperature (Tm) and aggregation temperature (Tagg): Tm is the temperature at which 50% of the protein components denature during the heating process, and Tag is the temperature at which the protein denatures. This is the temperature at which aggregation occurs during the temperature process. The sample was loaded into a Uni-tube and measured at a temperature rise from 25 to 95°C.

[0222] Tm and Taag measuring equipment: Uncle, the equipment manufacturer is Unchained.

[0223] SEC size exclusion chromatography This is an analytical method that separates solutes based on the correlation between the pore size of gel pores and the coil size of polymer sample molecules.

[0224] SEC% (percentage of SEC monomer) = Amonomer / Atotal × 100% (Amonomer is the peak area of ​​the main peak monomer in the sample, and Atotal is the sum of all peak areas). ΔSEC% = SEC% of formulation after stability test - SEC% of formulation before stability test.

[0225] SEC measurement equipment: Agilent HPLC1260.

[0226] Column: Tosoh, TSKgel G3000SWXL (7.8 mm × 30 cm, 5 μm).

[0227] NR-CE capillary gel electrophoresis This is electrophoresis, which is carried out by moving a gel as a support medium in a capillary, and is a method of separating samples according to their molecular weight at a constant voltage.

[0228] NR-CE% (purity percentage of non-reduced CE) = A main peak / A total × 100% (A main peak is the peak area of ​​the main peak in the sample, and A total is the sum of all peak areas.) ΔNR-CE% = NR-CE% of formulation after stability test - NR-CE% of formulation before stability test.

[0229] Equipment for CE measurement: Beckman capillary electrophoresis apparatus, model number PA800 plus.

[0230] iCIEF Imaging Capillary Isoelectric Focusing A capillary is used as a separation channel, and a DC voltage is applied to both ends of the capillary. The ampholyte solution in the capillary forms a pH gradient over a certain range, and each component migrates to its respective isoelectric point based on the difference in charge and is focused into a very narrow compartment, thereby achieving separation of the components.

[0231] icIEF% (percentage of main peak content) = A main peak area / A total area × 100% (A total area is the sum of the areas of the acidic peak, main peak and basic peak). ΔicIEF% = icIEF% of formulation after stability test - icIEF% of formulation before stability test.

[0232] Equipment for icIEF measurement: Protein Simple, model number Maurice.

[0233] The RANKL-NGF bispecific antibody used in the following formulation preparation examples is the above-mentioned bispecific antibody 1, sometimes abbreviated as "antibody."

[0234] Preparative Example 1. pH Screening of RANKL-NGF Bispecific Antibody Formulations A 10 mM citrate-disodium hydrogen phosphate buffer solution was used, with ten different pH settings: 4.2, 4.6, 5.0, 5.4, 5.8, 6.2, 6.6, 7.0, 7.4, and 7.8. Ten RANKL-NGF bispecific antibody formulations were prepared at 20 mg / mL. The melting temperature (Tm) of the samples was measured to examine the conformational stability of the antibody at different pH conditions. The aggregation temperature (Tag) of the samples was also measured to examine the colloidal stability of the antibody at different pH conditions. The thermal stability of the antibody at different pH conditions was also examined by measuring the appearance, SEC, and NRCE of the samples at 40°C.

[0235] 1) 10 mM citrate-disodium hydrogen phosphate, pH 4.2; 2) 10 mM citrate-disodium hydrogen phosphate, pH 4.6; 3) 10 mM citrate-disodium hydrogen phosphate, pH 5.0; 4) 10 mM citrate-disodium hydrogen phosphate, pH 5.4; 5) 10 mM citric acid-disodium hydrogen phosphate, pH 5.8; 6) 10 mM citric acid-disodium hydrogen phosphate, pH 6.2; 7) 10 mM citric acid-disodium hydrogen phosphate, pH 6.6; 8) 10 mM citrate-disodium hydrogen phosphate, pH 7.0; 9) 10 mM citric acid-disodium hydrogen phosphate, pH 7.4; 10) 10 mM citric acid-disodium hydrogen phosphate, pH 7.8.

[0236] [Table 17] [Table 18] [Table 19]

[0237] The results showed that the RANKL-NGF bispecific antibody had higher Tm and Tagg values ​​at pH 4.6-5.4, indicating better conformational and colloidal stability of the antibody in this pH range. After one week of observation at high temperature (40°C), the appearance of the formulation in the pH range of 4.2-5.4 showed a small number of visible protein particles, while large amounts of protein particles were observed at other pH conditions. In terms of purity, significant protein aggregation was observed after 7 days of incubation at 40°C in the pH 4.2, 7.4, and 7.8 systems. A small amount of aggregation was observed at pH 7.0, but no significant aggregation was observed in any of the other pH systems, and no significant differences were observed between groups. According to the NRCE results, the protein in the pH 4.6-5.8 system had a relatively low fragment content at TO, of which the fragment content at pH 5.0-5.8 after 7 days of incubation at 40°C was less than 4%. Summarizing the molecular properties, appearance and SEC results of the above samples, the antibody expression was best in the pH 4.6-5.4 system, so the intermediate pH value of 5.0 was selected as the preferred pH condition for further research.

[0238] Preparative Example 2. Screening of buffer systems for RANKL-NGF bispecific antibody formulations Three buffer systems were selected: 10 mM acetic acid / sodium acetate at pH 4.8 and pH 5.2, and 10 mM histidine / histidine hydrochloride at pH 5.2. 75 mg / mL sucrose was used as a stabilizer. A total of three formulation samples were prepared. The RANKL-NGF bispecific antibody concentration was 20 mg / mL. The optimal buffer system was screened by examining the sample appearance, SEC-HPLC, NRCE, and iCIEF purity. The three formulations were then incubated at high temperature (40°C) for 2 weeks, at 25°C for 2 and 4 weeks, at 2-8°C for 2 and 4 weeks, and freeze-thawed three and five times (-35°C and room temperature).

[0239] 1) 10 mM acetic acid-sodium acetate, pH 4.8, 75 mg / mL sucrose, 20 mg / mL antibody; 2) 10 mM acetic acid-sodium acetate, pH 5.2, 75 mg / mL sucrose, 20 mg / mL antibody; 3) 10 mM histidine-histidine hydrochloride, pH 5.2, 75 mg / mL sucrose, 20 mg / mL antibody.

[0240] [Table 20] [Table 21] [Table 22] [Table 23]

[0241] The results showed that after two weeks of incubation at 25°C and 40°C, the protein in the 10mM acetic acid-sodium acetate pH 5.2 system exhibited opalescence, while the protein in the 10mM acetic acid-sodium acetate pH 4.8 system exhibited slight opalescence. A small amount of protein particles appeared in both formulations. After two weeks of incubation at 40°C, the protein in the 10mM histidine-histidine hydrochloride pH 5.2 system exhibited a large amount of protein particles. Visual analysis indicated that the protein was more stable in the 10mM acetic acid-sodium acetate buffer system. In terms of purity, the percentages of polymer, monomer, and fragments in each group of formulations did not change significantly under different stability conditions compared to TO, and there was no significant difference between the groups, indicating superior purity and stability measured by SEC-HPLC. At high temperatures, the increase in NRCE fragments in the 10mM histidine-histidine hydrochloride system was more significant than in the acetate buffer system. iCIEF detection showed no significant differences between the formulations. As described above, a 10 mM acetic acid-sodium acetate buffer system was selected for the subsequent formulation development.

[0242] Preparation Example 3. RANKL-NGF Bispecific Antibody Formulation Concentration and Stabilizer Screening To ensure a better pH buffering effect, the buffer concentration was increased to 20 mM. 20 mM acetic acid-sodium acetate was selected as the buffer system, pH 5.0 was selected, 0.2 mg / mL polysorbate 80 was selected as the surfactant, 75 mg / mL sucrose, 240 mM proline, or 0.8% (w / v) sodium chloride was selected as the stabilizer, and the antibody concentrations were 20 mg / mL, 50 mg / mL, and 70 mg / mL, respectively. A total of five formulation samples were prepared, and the stability of the samples was examined under shaking (300 rpm / 25°C), repeated freeze-thawing (-35°C / room temperature), high temperature (40°C), 25°C, and light exposure conditions.

[0243] 1) 20 mM acetic acid-sodium acetate, pH 5.0, 75 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 50 mg / mL antibody; 2) 20 mM acetic acid-sodium acetate, pH 5.0, 240 mM proline, 0.2 mg / mL polysorbate 80, 50 mg / mL antibody; 3) 20 mM acetic acid-sodium acetate, pH 5.0, 0.8% (w / v) sodium chloride, 0.2 mg / mL polysorbate 80, 50 mg / mL antibody; 4) 20 mM acetic acid-sodium acetate, pH 5.0, 75 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 20 mg / mL antibody; 5) 20 mM acetic acid-sodium acetate, pH 5.0, 75 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 70 mg / mL antibody.

[0244] [Table 24] [Table 25] [Table 26] [Table 27]

[0245] The results showed that compared with the other formulations, formulation 3 had a slightly heavier opalescence and a slightly inferior appearance. In terms of purity, the SEC-HPLC polymer and NRCE fragment contents of each formulation did not change significantly under the conditions of shaking, light exposure, repeated freeze-thawing, and 25°C. At a higher temperature of 40°C, the SEC-HPLC polymer content of formulation 3 increased significantly, indicating poorer stability of this formulation. The NRCE fragment content of formulation 2 increased less, indicating that proline can be used as a stabilizer to significantly improve antibody stability and that the higher the antibody concentration, the higher the NRCE purity (the difference between the 50 mg / mL and 70 mg / mL formulations was relatively small). For each formulation, the iCIEF acidic, neutral, and basic peaks did not change significantly under the conditions of shaking, repeated freeze-thawing, and 25°C. However, after 4 weeks at a high temperature of 40°C, the acidic peaks all increased. Formulation 2 had the highest main peak content, and the difference between the 50 mg / mL and 70 mg / mL formulations was relatively small. To summarize the results, proline was selected as the stabilizer, the antibody concentration was 70 mg / mL, and the pH was 5.0.

[0246] Preparative Example 4. Screening of surfactant concentration in RANKL-NGF bispecific antibody formulations A 20 mM acetic acid-sodium acetate, pH 5.0 buffer system was selected, 240 mM proline was used as a stabilizer, the RANKL-NGF bispecific antibody concentration was 70 mg / mL, and the polysorbate 80 concentrations were 0.1 mg / mL, 0.2 mg / mL, and 0.4 mg / mL, respectively. Three groups of formulation samples were prepared, and the stability of the samples was examined under shaking (300 rpm / 25°C), repeated freeze-thawing (-35°C / room temperature), and high temperature (40°C) conditions.

[0247] 1) 20 mM acetic acid-sodium acetate, pH 5.0, 240 mM proline, 0.1 mg / mL polysorbate 80, 70 mg / mL antibody; 2) 20 mM acetic acid-sodium acetate, pH 5.0, 240 mM proline, 0.2 mg / mL polysorbate 80, 70 mg / mL antibody; 3) 20 mM acetic acid-sodium acetate, pH 5.0, 240 mM proline, 0.4 mg / mL polysorbate 80, 70 mg / mL antibody.

[0248] [Table 28] [Table 29] [Table 30] [Table 31]

[0249] The results showed that the polymer, fragment, and acidic / basic peak contents all tended to increase with increasing polysorbate 80 concentration, and the antibody stability was relatively poor when the polysorbate 80 concentration was 0.4 mg / mL. Therefore, the polysorbate 80 concentration is preferably 0.1 mg / mL.

[0250] Preparative Example 5. Preferred Formulation Stability Studies of RANKL-NGF Bispecific Antibody A 20 mM acetic acid-sodium acetate, pH 5.0 buffer system was selected, and 240 mM proline was used as a stabilizer. A RANKL-NGF bispecific antibody concentration of 70 mg / mL and a polysorbate 80 concentration of 0.1 mg / mL were prepared, and the stability of the formulation was examined under accelerated conditions (25±2°C / 60% RH±5RH) and long-term conditions (2-8°C).

[0251] [Table 32]

[0252] Note: NT indicates not detected.

[0253] [Table 33]

[0254] Note: NT indicates not detected.

[0255] The results showed that after 6 months of storage under accelerated conditions (25±2°C / 60%RH±5RH), the iCIEF main peak decreased by approximately 12.7%, with no significant changes in other detection parameters. However, after 12 months of storage under long-term conditions (2-8°C), none of the detection parameters changed significantly compared to time 0, demonstrating the excellent long-term stability of the RANKL-NGF bispecific antibody formulation.

[0256] Preparation Example 6. Alternative formulation methods The present disclosure provides a RANKL-NGF bispecific antibody drug formulation comprising "50 to 77 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 4.6 to 5.4," (1) 70 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 4.6, (2) 70 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 5.0; (3) 70 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 5.4; (4) 50 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 4.6; (5) 50 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 5.0; (6) 50 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 5.4; (7) 77 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 4.6; (8) 77 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 5.0, (9) 77 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 5.4, but are not limited to these.

[0257] The experimental results showed that all of the RANKL-NGF bispecific antibody formulations prepared by the above formulation methods had excellent stability and were applicable to the preparation of RANKL-NGF bispecific antibody drugs.

Claims

1. A pharmaceutical composition comprising an anti-RANKL-NGF bispecific antibody and a buffer, the anti-RANKL-NGF bispecific antibody comprises a first antigen-binding domain that specifically binds to RANKL and a second antigen-binding domain that specifically binds to NGF; the buffer is an acetate buffer, a histidine buffer, or a phosphate buffer; Preferably, the buffer is an acetic acid-sodium acetate buffer, a histidine-histidine hydrochloride buffer, or a citric acid-disodium hydrogen phosphate buffer; More preferably, the buffer is an acetic acid-sodium acetate buffer or a histidine-histidine hydrochloride buffer; Most preferably, the buffer is an acetic acid-sodium acetate buffer. Pharmaceutical compositions.

2. the pH of the pharmaceutical composition is 4.2 to 7.0; Preferably, the pH of the pharmaceutical composition is 4.6 to 5.4; More preferably, the pH of the pharmaceutical composition is 4.8 to 5.

2. The pharmaceutical composition of claim 1.

3. the concentration of the anti-RANKL-NGF bispecific antibody is 1 to 150 mg / mL; Preferably, the concentration of the anti-RANKL-NGF bispecific antibody is 10 to 80 mg / mL; More preferably, the concentration of the anti-RANKL-NGF bispecific antibody is 63 to 77 mg / mL. The pharmaceutical composition according to claim 1 or 2.

4. the pharmaceutical composition comprises a surfactant; Preferably, the surfactant is a polysorbate or a poloxamer; More preferably, the surfactant is polysorbate 20 or polysorbate 80; Most preferably, the surfactant is polysorbate 80. The pharmaceutical composition according to any one of claims 1 to 3.

5. the surfactant has a concentration of 0.01 to 1.0 mg / mL; Preferably, the concentration of the surfactant is 0.01 to 0.6 mg / mL; More preferably, the concentration of the surfactant is 0.01 to 0.2 mg / mL; Most preferably, the concentration of the surfactant is 0.05 to 0.15 mg / mL. The pharmaceutical composition according to claim 4.

6. the pharmaceutical composition comprises a stabilizer; Preferably, the stabilizer is one or more selected from the group consisting of proline, sucrose, trehalose, sorbitol, arginine, glycine and sodium chloride; More preferably, the stabilizer is proline, sucrose or sodium chloride; Most preferably, the stabilizer is proline. The pharmaceutical composition according to any one of claims 1 to 5.

7. The concentration of the stabilizer is 1 to 300 mM; Preferably, the concentration of the stabilizer is 25 to 290 mM; More preferably, the concentration of the stabilizer is 210 to 270 mM. The pharmaceutical composition according to claim 6.

8. The concentration of the buffer is 5 to 100 mM; Preferably, the concentration of the buffer is 10 to 50 mM; More preferably, the concentration of the buffer is 10 to 30 mM; Most preferably, the concentration of the buffer is 16-24 mM. The pharmaceutical composition according to any one of claims 1 to 7.

9. The anti-RANKL-NGF bispecific antibody comprises: at least one first antigen-binding domain that specifically binds to RANKL; and at least one second antigen-binding domain that specifically binds to NGF; Preferably, The anti-RANKL-NGF bispecific antibody comprises: two first antigen-binding domains that specifically bind to RANKL; and two second antigen-binding domains that specifically bind to NGF; More preferably, the anti-RANKL-NGF bispecific antibody has the structure shown in FIG. The pharmaceutical composition according to any one of claims 1 to 8.

10. In the anti-RANKL-NGF bispecific antibody, the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; Preferably, In the anti-RANKL-NGF bispecific antibody, the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO:

8. The pharmaceutical composition of claim 9.

11. In the anti-RANKL-NGF bispecific antibody, the second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; and the light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 20; Preferably, In the anti-RANKL-NGF bispecific antibody, the second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22; More preferably, In the anti-RANKL-NGF bispecific antibody, the second antigen-binding domain that specifically binds to NGF comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 24 and the light chain comprises the amino acid sequence of SEQ ID NO:

25. The pharmaceutical composition according to claim 9 or 10.

12. The following ingredients: (a) 1 to 150 mg / mL of the anti-RANKL-NGF bispecific antibody; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 1 to 300 mM of a stabilizer; (d) 5 to 100 mM of a buffering agent, wherein the pH of the pharmaceutical composition is 4.2 to 7.0; Preferably, the pharmaceutical composition comprises the following ingredients: (a) 10 to 80 mg / mL of the anti-RANKL-NGF bispecific antibody; (b) 0.01 to 0.6 mg / mL of polysorbate 20 or polysorbate 80; (c) 25 to 290 mM proline; (d) 10-50 mM acetate buffer, wherein the pH of the pharmaceutical composition is 4.6-5.4; More preferably, the pharmaceutical composition comprises the following ingredients: (a) 63 to 77 mg / mL of an anti-RANKL-NGF bispecific antibody; (b) 0.01 to 0.2 mg / mL of polysorbate 80; (c) 210-270 mM proline; (d) 10 to 30 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.2; Most preferably, the pharmaceutical composition comprises the following ingredients: (a) 63 to 77 mg / mL of an anti-RANKL-NGF bispecific antibody; (b) 0.05 to 0.15 mg / mL of polysorbate 80; (c) 210-270 mM proline; (d) 16 to 24 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.

2. The pharmaceutical composition according to any one of claims 1 to 11.

13. Once reconstituted, it can form a pharmaceutical composition according to any one of claims 1 to 12. Lyophilized formulation.

14. 13. The method of claim 1, further comprising the step of lyophilizing the pharmaceutical composition of claim 1. Methods for preparing lyophilized formulations.

15. Obtained by the method according to claim 14 Lyophilized formulation.

16. a subcutaneous injection formulation, an intravenous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation, preferably a subcutaneous injection formulation; The pharmaceutical composition according to any one of claims 1 to 12.

17. A method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 12 or the lyophilized formulation according to claim 13 or 15; Preferably, the disorder is pain, joint stiffness or bone loss; More preferably, the pain is selected from osteoarthritic pain, rheumatoid arthritis pain, gout, bone cancer pain, fracture pain, post-operative pain, cancer pain, bladder pain syndrome, musculoskeletal pain, prostatitis, pelvic pain, interstitial cystitis, lower back pain, dysmenorrhea, pain associated with bone disease, trigeminal neuralgia, post-herpetic neuralgia, herpes zoster infection, sciatica, migraine, diabetic neuropathy and peripheral nerve associated pain, wherein the bone loss is associated with at least one condition selected from the group consisting of osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, bone loss, osteoporosis, osteonecrosis, bone damage, bone resorption, osteogenesis imperfecta, inflammation, autoimmune disease, enteritis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, periodontal bone resorption, osteolytic metastasis, and cancer; method.

18. The cancer is selected from breast cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, esophageal cancer, rectal cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, melanoma, multiple myeloma, osteosarcoma, lymphoma, non-small cell lung cancer, bone tumor, and Hodgkin's disease; 18. The method of claim 17.

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